Counting biomolecules using single-molecule imaging technique*

Quantifying the protein expression level and detecting aberrant proteins are critical to many therapeutic areas such as cancer development, brain injury, inflammation and infectious diseases. However, it is difficult to detect a small trace of the proteins as contrasted with other biomolecules such as DNA or RNA. It is even more challenging to reveal their oligomerization state which is often a hallmark of several diseases. Here, we demonstrate quantitative analysis of endogenous proteins from human brain tissue using a single-molecule pull-down assay. Single-molecule imaging technique enables us to directly count the number of single proteins and reveal the stoichiometry of the protein complex. By utilizing in vivo crosslinker, we can preserve the native oligomerization state of proteins. Quantitative labeling and single-molecule intensity analysis allow to precisely count the number of proteins. Our technique is a powerful diagnostic tool for the detection of various neurodegenerative diseases, such as Parkinson’s disease and Alzheimer’s disease, and can be used to analyze various biospecimens.